SUMMARY PhotoSound Technologies, Inc. proposes to develop a novel imaging modality for characterization and pre- clinical research of murine models. The technology will be capable of three-dimensional functional and molecular imaging of fluorescent labels and reporter genes mapped with high fidelity over robust anatomical structures, such as skin, central and peripheral vasculature, and internal organs. The developed instrument could be used in broad spectrum of pre-clinical research including cancer, toxicology, tissue engineering and regeneration, cardiovascular and developmental biology. Optical in vivo imaging methods (fluorescence and bioluminescence) found great popularity among researchers as affordable, convenient, and very sensitive molecular imaging tools for pre-clinical studies and development of animal models. However, their stand-alone application is impeded by poor spatial resolution and limitations imposed by two-dimensionality of the images. A high-resolution in vivo 3D imaging method, which could be easily integrated with optical imaging in a single instrument, would have a great impact on the entire field of small animal research. Photoacoustic tomography is an emerging biomedical imaging modality that has all those requirements: (1) 150-500 m resolution of 3D whole body images; (2) Ability to use the same instrumentation for excitation of fluorescence and generation of photoacoustic effect; (3) 3D scans in less than 1 minute. However, its in vivo sensitivity to detection of fluorophores is inferior as compared to regular fluorescence techniques. Our proposal is based on pioneering co-registered integration of fluorescence and photoacoustic modalities in a single compact 3D configuration (PAFT-3D) defeating shortcomings of each individual technology. The Phase I project is organized in three specific aims assessing feasibility of: (1) Technological implementation for co-registered 3D photoacoustic tomography and fluorescence in a single compact instrument for imaging live anesthetized mice; (2) Imaging fluorophores and anatomical structures of mice with resolution exceeding that of established fluorescence imaging; (3) Providing imaging information valuable for development and characterization of pre-clinical animal models with case study focused on murine models of metastatic breast cancer. Phase II will be focused on development and in vivo validation of a commercial instrument with sensitivity and imaging procedures optimized for various areas of animal model development and preclinical research. Ultimate commercial system will enable in vivo visualization and analysis of native hemoglobin, fluorophores, nanoparticles, and other photosensitive constructs used for tracking, mapping, and longitudinal studies. The value proposition of the PAFT-3D will be built not only around uniqueness and technological superiority of the product, but also by making it affordable for a broad population of research groups and centers.